T lymphocytes are cells of the adaptive immune system that mainly develop in the thymus and are selected to express a unique and exquisitely-specific T-cell receptor (TCR) that will both remain tolerant (non-responsive) to self antigens, and yet bet able to recognize and respond to a foreign linear peptide in the context of self-major histocompatibility complex (MHC) molecules.
During differentiation, progenitors to T cells arise from stem cells in the bone marrow, and are then recruited to the thymus, entering via vessels in the perimedullary cortex, where they begin differentiation into T cells. Following a series of ligand/receptor and cytokine or chemokine/receptor interactions, the initial “double negative” thymocytes progress through maturation, eventually rearranging T-cell receptor genes to generate a productive TCRβ chain gene. The TCRβ chain protein is coexpressed with the pre-T alpha receptor, and this triggers co-expression of both the CD4 and CD8 coreceptors by cells that are referred to as double positive thymocytes (DPs). Upon a productive TCRα rearrangement, the TCRβ and TCRα chain proteins are coexpressed as a heterodimer in association with proteins of the CD3 complex. Positive selection then occurs, resulting in the upregulation of the surface levels of the αβ TCR/CD3 complex and loss of either CD8 to form CD4+CD8− thymocytes (MHC class II selection), or loss of CD8 to form CD4−CD8+ thymocytes (MHC class I selection). These single-positive thymocytes move into the medulla where negative selection occurs to purge the repertoire of autoreactive T cells.
Following selection, these naïve T cells are exported from the thymus. The rate of thymic export appears to remain constant throughout the lifetime of the individual but the volume of export is proportional to the volume of residual thymic mass index. Therefore, as individuals age, their thymus involutes and is largely converted into fat, and there is a concomitant decrease in the number of recent thymic emigrants that are exported on a daily basis. Despite this reduction in thymic output throughout the time course of life in individuals, the absolute number of T cells in the peripheral T-cell pool remains surprisingly constant throughout life.
Following thymic development, CD4+ and CD8+ T cells are exported into the peripheral circulation as naïve T cells, which survey self-major histocompatibility complex (MHC) molecules for foreign peptide antigens. In response to cognate antigen, they differentiate into effector and memory T lymphocytes. The developmental stage of human T-cell maturation following export from the thymus has not been extensively characterized. A limited number of studies in other species have suggested that recent thymic emigrants (RTEs) are not fully mature and require a developmental period of post-thymic maturation to become fully functional T cells. In the rat, lack of both RT6 and CD45RC expression distinguish RTEs from the rest of the mature T cell pool. Unfortunately, human homologs for markers such as RT6 or CD45RC have not been identified for humans, and experimental approaches useful in animals, such as ectopic GFP expression or intrathymic injections of dyes, are not possible. Therefore, aspects of human post-thymic T cell development remain unknown.
In order to overcome these challenges, investigators have developed a surrogate marker for RTE frequency in human T cell populations called sjTREC content analysis. sjTRECs, or signal joint T-cell receptor excision circles, are the signal joint byproducts of VDJ recombination leftover following TCR rearrangement in the thymus. Because they lack an origin of replication, they are not replicated during each cell division, and as such are diluted in T cells following cell proliferation. Relative or absolute TREC content can be used to determine the relative “age” of bulk populations of cells, and to roughly estimate the contribution of thymic output to the T-cell pool. However, TRECs can only be detected with a PCR assay that requires killing the cells; the TREC assay cannot determine at a single cell level whether a T cell is an RTE; and because a TREC has to be split between two daughter cells, even TREC+ naïve T cells may have undergone some rounds of division. TRECs are also assumed to be equally stable in various naïve T-cell populations in the absence of cell proliferation, but whether this is actually the case remains unknown.
A unique cell-surface marker that directly detects naïve T cells, particularly live RTEs, at the single cell level is highly desirable for enumerating these cells and understanding their function. The ready identification of these cells in humans and an understanding of their maturation and functional attributes are important not only for understanding the basic biology of T-cell development and function, but also for the development of better vaccines for human neonates and infants. Furthermore, such a marker would allow one to monitor the kinetics of thymic output in normal patients over the adult lifespan, and T-cell reconstitution in those patients with transient deficiencies in CD4+ T cells, such as those with HIV-infection, hematopoietic stem cell transplantation, or chemotherapy treatment, and those with inherited immunodeficiencies involving T cells, such as severe combined immunodeficiency (SCID).